Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

Cyclin D1 overexpression permits the reproducible detection of senescent human vascular smooth muscle cells.

The senescence of mitotic cells is hypothesized to play a causal role in organismal aging. Cultures of normal human cells become senescent in vitro as a result of a continuous decline in the mitotic fraction from cell turnover. However, one potential barrier to the evaluation of the frequency and distribution of senescent cells in tissues is the absence of a panel of robust markers for the senescent state. In parallel with an analysis of the growth kinetics of human vascular smooth muscle cells, we have undertaken transcriptomic comparisons of early- and late-passage cultures of human vascular smooth muscle cells to identify potential markers that can distinguish between senescent and growth-competent cells. A wide range of genes are upregulated at senescence in human vascular smooth muscle cells. In particular, we have identified a 12-fold upregulation of expression in the cyclin D1 message, which is reflected in a concomitant upregulation at the protein level. Quantitative cytochemical analysis of senescent and growing vascular smooth muscle cells indicates that cyclin D1 reactivity is a considerably better marker of replicative senescence than senescence-associated beta-galactosidase activity. We have applied this new marker (in combination with Ki67, COMET, and TUNEL staining) to the study of human vascular smooth muscle cells treated with resveratrol, a putative anti-aging molecule known to have significant effects on cell growth.

Surgical-site infection rates and risk factor analysis in coronary artery bypass graft surgery.

BACKGROUND: The Victorian Infection Control Surveillance Project (VICSP) is a multicenter collaborative surveillance project established by infection control practitioners. Five public hospitals contributed data for patients undergoing coronary artery bypass graft (CABG) surgery. OBJECTIVE: To determine the aggregate and comparative interhospital surgical-site infection (SSI) rates for patients undergoing CABG surgery and the risk factors for SSI in this patient group. METHOD: Each institution used standardized definitions of SSI, risk adjustment, and reporting methodology according to the National Nosocomial Infections Surveillance System of the Centers for Disease Control and Prevention. Data on potential risk factors were prospectively collected. RESULTS: For 4,474 patients undergoing CABG surgery, the aggregate SSI rate was 7.8 infections per 100 procedures (95% confidence interval [CI95], 7.0-8.5), with individual institutions ranging between 4.5 and 10.7 infections per 100 procedures. Multivariate risk factor analysis demonstrated age (odds ratio [OR], 1.02; CI95, 1.01-1.04; P < .001), obesity (OR, 1.8; CI95, 1.4-2.3; P < .001), and diabetes mellitus (OR, 1.6; CI95, 1.2-2.1; P < .001) as independent predictors of SSI. Three hundred thirty-four organisms were isolated from 296 SSIs. Of the total SSIs, methicillin-resistant Staphylococcus aureus was isolated from 32%, methicillin-sensitive S. aureus from 24%, gram-negative bacilli (eg, Enterobacter and Escherichia coli) from 18%, and miscellaneous organisms from the remainder. CONCLUSION: We documented aggregate and comparative SSI rates among five Victorian public hospitals performing CABG surgery and defined specific independent risk factors for SSI. VICSP data offer opportunities for targeted interventions to reduce SSI following cardiac surgery.